The Aims of the parent grant to this requested revision supplement are to test whether genetic inactivation of immunosuppressive A2 adenosine receptor subtypes A2A and A2B (A2AR and A2BR, respectively) will prevent the inhibition of anti-tumor T cells by the tumor-produced extracellular adenosine. This was expected to facilitate the complete rejection of tumors by antitumor T cells in mice. The Parent grant was to determine the limits of improvements of anti-tumor T cell activities by complete elimination of genes for A2AR and A2BR. We have established that the genetic elimination of A2AR-but not of A2BR-strongly improves tumor rejection. In this revised application, we propose to expand the scope, aims, design, and methods of the current grant and render novel immunotherapies of cancer more effective by pharmacologically inactivating A2AR. This proposal is designed to compare the effects of synthetic and natural A2AR antagonists on activities of cancer vaccine-induced and adoptively-transferred anti-tumor T cells and tumor rejection. This request is also appropriate to achieve Recovery Act goals since it results in hiring and retaining scientists, students and animal care workers, adds another PI, and accelerates the translation of novel and promising cancer immunotherapy into clinical therapy. Indeed, in our preliminary studies we demonstrated complete tumor rejection and survival due to the combination of anti-tumor T cells with A2AR antagonist. This convinced us and clinical cancer immunologists at the Dana Farber Cancer Institute to prepare clinical trials using a combination of existing cancer immunotherapy protocols with A2AR antagonists. However, there is still a large body of preclinical in vitro and in vivo experiments to be done in order to develop a better understanding of biochemical, cellular immunological and pharmacological effects of A2AR antagonists likely to be tested in humans. Therefore, we propose to test different selective synthetic and natural (e.g. caffeine and theophylline) A2AR antagonists in preclinical models of cancer vaccines and adoptive immunotherapy. Importantly, the A2AR antagonist KW-6002, developed for treatment of Parkinson Disease, was found to be safe in Phase III trials, and it shows very strong anti-tumor activity in our studies when combined with anti-tumor T cells. PUBLIC HEALTH RELEVANCE: In this revised application, we propose to significantly expand the scope of the current grant to render novel immunotherapies of cancer more effective by pharmacologically preventing the inhibition of anti-tumor T cells by tumor-produced adenosine. We will use novel synthetic and natural A2AR antagonists, including caffeine and theophylline, together with cancer vaccine-induced or with adoptively-transferred anti-tumor T killer cells. This research results in hiring and retaining scientists, students and animal care workers, adds another PI, and accelerates the translation of novel and promising cancer immunotherapy into clinic therapy.